Solanidine is a sensitive and specific dietary biomarker for CYP2D6 activity

Background Individual assessment of CYP enzyme activities can be challenging. Recently, the potato alkaloid solanidine was suggested as a biomarker for CYP2D6 activity. Here, we aimed to characterize the sensitivity and specificity of solanidine as a CYP2D6 biomarker among Finnish volunteers with known CYP2D6 genotypes. Results Using non-targeted metabolomics analysis, we identified 9152 metabolite features in the fasting plasma samples of 356 healthy volunteers. Machine learning models suggested strong association between CYP2D6 genotype-based phenotype classes with a metabolite feature identified as solanidine. Plasma solanidine concentration was 1887% higher in genetically poor CYP2D6 metabolizers (gPM) (n = 9; 95% confidence interval 755%, 4515%; P = 1.88 × 10–11), 74% higher in intermediate CYP2D6 metabolizers (gIM) (n = 89; 27%, 138%; P = 6.40 × 10–4), and 35% lower in ultrarapid CYP2D6 metabolizers (gUM) (n = 20; 64%, − 17%; P = 0.151) than in genetically normal CYP2D6 metabolizers (gNM; n = 196). The solanidine metabolites m/z 444 and 430 to solanidine concentration ratios showed even stronger associations with CYP2D6 phenotypes. Furthermore, the areas under the receiver operating characteristic and precision–recall curves for these metabolic ratios showed equal or better performances for identifying the gPM, gIM, and gUM phenotype groups than the other metabolites, their ratios to solanidine, or solanidine alone. In vitro studies with human recombinant CYP enzymes showed that solanidine was metabolized mainly by CYP2D6, with a minor contribution from CYP3A4/5. In human liver microsomes, the CYP2D6 inhibitor paroxetine nearly completely (95%) inhibited the metabolism of solanidine. In a genome-wide association study, several variants near the CYP2D6 gene associated with plasma solanidine metabolite ratios. Conclusions These results are in line with earlier studies and further indicate that solanidine and its metabolites are sensitive and specific biomarkers for measuring CYP2D6 activity. Since potato consumption is common worldwide, this biomarker could be useful for evaluating CYP2D6-mediated drug–drug interactions and to improve prediction of CYP2D6 activity in addition to genotyping. Supplementary Information The online version contains supplementary material available at 10.1186/s40246-024-00579-8.

Then, the metabolic depletion of a low, clinically relevant concentration of solanidine (10 nM) was investigated in HLMs (0.2 mg/mL protein) and human recombinant CYP2D6 (0.1 mg/mL Bactosome protein) in the presence of NADPH (1 mM).Preincubation time was 5 min, and samples were taken at 0, 5, 15, 30, and 60 min.
Inhibition experiments were carried out in HLMs using paroxetine as a mechanism-based inhibitor of CYP2D6 and ritonavir as a mechanism-based inhibitor of CYP3A4/5. 1,2Due to time-dependency of the mechanism-based inhibition, paroxetine (1 µM) or ritonavir (0.1 µM) were first pre-incubated with HLMs (0.2 mg/mL) and NADPH (1 mM) for 30 min prior to addition of solanidine (0.1 µM) to the reaction mixtures.The inhibition by paroxetine was also investigated with a low solanidine concentration of 10 nM.Control samples were done without the inhibitors.Samples were taken at 0, 5, 15, 30, and 60 min.
To investigate the formation of solanidine metabolites other than OH-solanidine (m/z 414), additional experiments with higher concentrations of HLM (2 mg/mL) and solanidine (5 µM), and a longer incubation time (150 min) were carried out.Here, the NADPH RapidStart Regeneration System for Extended Metabolism (Xenotech) was used to regenerate NADPH to the reaction.These incubations were also done with and without paroxetine (1 µM) and ritonavir (0.1 µM).
Measured HLM CLint,u values were scaled to CLint,in vivo using 39.79 mg microsomal protein/g liver, and liver volume and density values of 1.65 l and 1,080 g/l liver (Simcyp Population-Based Simulator V20).In the final step, hepatic blood clearance (CLH) values were calculated using the well-stirred model. 3  =   ×  , ×  ,    +  , ×  ,  where QH is the hepatic blood flow (1.61 l/min) 4 and fu,B is the unbound fraction of solanidine in blood.fu,B was calculated according to  , =  , × 1  ⁄ , where fu,p and BP are the predicted unbound fraction in plasma (0.065) and blood-to-plasma concentration ratio (0.862) of solanidine, respectively (https://members.simcyp.com/account/tools/fumic/,accessed on Feb 9, 2023).The calculated fu,B equaled to 0.075.
The kinetics of solanidine depletion in the HLM depletion and inhibition experiments were analyzed using GraphPad Prism (version 7.03; GraphPad Software, Inc., San Diego, CA, USA).Depletion rate constants (kdep) were determined using linear regression of ln-transformed solanidine concentrations, and the intrinsic clearance (CLint) of solanidine was expressed as CLint = kdep/[M], where [M] is the HLM protein concentration used in the experiments (0.2 mg/mL).The change in solanidine CLint due to inhibition was calculated by comparing the CLint values in samples containing the inhibitors with the CLint value in the control samples.

Table S1 .
Demographic characteristics of study subjects

Table S3 .
CYP2D6 genotype distribution in the study population of 356 healthy Finnish volunteers.

Table S4 .
Associations of metabolite features found in non-targeted metabolomics analysis of human plasma with CYP2D6 phenotypes

Table S5 . Effect of CYP2D6 phenotype on plasma solanidine metabolites in healthy volunteers (n=314)
Metabolite concentrations are given in arbitrary units relative to the metabolite to internal standard peak area ratio.